Abstract

In this work, a simple method for alcohol synthesis with high enantiomeric purity
was proposed. For this, colloidal gold and silver surface modifications with 3-mercaptopropanoic
acid and cysteamine were used to generate carboxyl and amine functionalized gold and
silver nanoparticles of 15 and 45 nm, respectively. Alcohol dehydrogenase from Thermoanaerobium brockii (TbADH) and its cofactor (NADPH) were physical and covalent (through direct adsorption
and using cross-linker) immobilized on nanoparticles' surface. In contrast to the
physical and covalent immobilizations that led to a loss of 90% of the initial enzyme
activity and 98% immobilization, the use of a cross-linker in immobilization process
promoted a loss to 30% of the initial enzyme activity and >92% immobilization. The
yield of NADPH immobilization was about 80%. The best results in terms of activity
were obtained with Ag-citr nanoparticle functionalized with carboxyl groups (Ag-COOH),
Au-COOH(CTAB), and Au-citr functionalized with amine groups and stabilized with CTAB
(Au-NH2(CTAB)) nanoparticles treated with 0.7% and 1.0% glutaraldehyde. Enzyme conformation
upon immobilization was studied using fluorescence and circular dichroism spectroscopies.
Shift in ellipticity at 222 nm with about 4 to 7 nm and significant decreasing in
fluorescence emission for all bioconjugates were observed by binding of TbADH to silver/gold
nanoparticles. Emission redshifting of 5 nm only for Ag-COOH-TbADH bioconjugate demonstrated
change in the microenvironment of TbADH. Enzyme immobilization on glutaraldehyde-treated
Au-NH2(CTAB) nanoparticles promotes an additional stabilization preserving about 50% of
enzyme activity after 15 days storage. Nanoparticles attached-TbADH-NADPH systems
were used for enantioselective (ee > 99%) synthesis of (S)-7-hydroxy-2-tetralol.